Project Summary Tonotopy is the most fundamental organizing principle of the vertebrate auditory system, meaning neurons of the auditory pathway are topographically arranged by their responses to different sound frequencies. It allows animals to separate a complex sound to its frequency components, forming the basis for sound discrimination. Despite its importance in auditory function, very little is known about the mechanisms that govern the formation of tonotopy in the auditory system. Knowing how auditory neurons generate tonotopic maps to process sound information, is, therefore, crucial for understanding auditory function and dysfunction such as central auditory processing disorders. To begin to elucidate the mechanisms that underlie tonotopic map formation, we are investigating how spiral ganglion neurons (SGNs) in the cochlea project their central fibers to target neurons in the cochlear nucleus (CN) with a tonotopic arrangement. Preliminary observations suggest that high- and low- frequency SGN fibers use distinct strategies during innervation of the CN. The birthdates of auditory neurons also influence their tonotopic location. To explore the molecular events underlying tonotopy, we have begun identifying axon targeting cues, including Ephs and ephrins that are expressed in the CN. Using a cochlear explant system, we found that a subset of auditory nerve fibers can be repelled by ephrin molecules. Based on these observations, we hypothesize that functionally distinct auditory neurons in the cochlea and CN use various strategies to establish connectivity and acquire positional identity during tonotopic map formation and they employ ephrin-Eph signaling as a molecular guidance mechanism to establish the tonotopic map. To test these hypotheses, we have three major aims: 1) we will use the Ngn1-CreERT2 mouse line to genetically label SGNs that respond to different sound frequencies to determine whether functionally distinct SGN populations employ different cellular strategies to target and innervate CN neurons during tonotopic map formation; 2) using a thymidine analog incorporation assay, we will examine whether CN neurons are born in a tonotopic gradient according to birthdates; 3) we will use cochlear explants and in situ hybridization to test whether ephrin/Eph forward signaling may play a role in tonotopic map formation in the CN. Results from these studies will provide novel insights into the cellular and molecular basis of how tonotopic maps are formed and allow us to better understand how disruption of tonotopy results in auditory dysfunction.